Methods to control protein heterogeneity

ABSTRACT

The instant invention relates to the field of protein production, and in particular to compositions and processes for controlling and limiting the heterogeneity of proteins expressed in host cells.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 14/958,211, filed Dec. 3, 2015, which is adivisional application of U.S. application Ser. No. 13/804,220, filedMar. 14, 2013, which claims priority to U.S. Provisional Application No.61/696,219, filed on Sep. 2, 2012, the disclosure of each of which areincorporated by reference herein in their entirety.

1. INTRODUCTION

The instant invention relates to the field of protein production, and inparticular to compositions and processes for controlling and limitingthe heterogeneity of proteins expressed in host cells.

2. BACKGROUND OF THE INVENTION

The production of proteins for biopharmaceutical applications typicallyinvolves the use of cell cultures that are known to produce proteinsexhibiting varying levels of heterogeneity. The basis for suchheterogeneity includes, but is not limited to, the presence of distinctglycosylation substitution patterns. For example, such heterogeneity canbe observed in increases in the fraction of proteins substituted withagalactosyl fucosylated biantennary oligosaccharides NGA2F+NGA2F-GlcNAcand decreases in the fraction of proteins substituted withgalactose-containing fucosylated biantennary oligosaccharides NA1F+NA2F.Such heterogeneity can be assayed by releasing oligosaccharides presenton the protein of interest via enzymatic digestion with N-glycanase.Once the glycans are released, the free reducing end of each glycan canbe labeled by reductive amination with a fluorescent tag. The resultinglabeled glycans are separated by normal-phase HPLC (NP-HPLC) anddetected by a fluorescence detector for quantitation.

Technological advances in recombinant protein production analysis haveprovided unique opportunities for identifying the extent ofheterogeneity exhibited by a particular protein population, particularlyin the context of large-scale production of recombinant proteins.Although such advances have allowed for the robust characterization ofprotein heterogeneity, there remains a need in the art to identifyculture conditions and production methods that allow for control overthe development of such heterogeneity. Control of protein heterogeneityis particularly advantageous in the context of cell culture processesused for commercially produced recombinant bio-therapeutics as suchheterogeneity has the potential to impact therapeutic utility. Theinstant invention addresses this need by providing compositions andprocesses to control protein heterogeneity.

3. SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods thatcontrol (modulate or limit) protein heterogeneity arising in apopulation of proteins, e.g., in the context of recombinant proteinproduction.

In certain embodiments, the heterogeneity corresponds to theglycosylation state of individual members of a population of proteins.In certain embodiments, control is exerted over the type ofglycosylation substitutions present on individual members of apopulation of proteins. In certain embodiments, control is exerted overthe extent of glycosylation substitutions present on individual membersof a population of proteins. In certain embodiments, control is exertedover both the type and extent of glycosylation substitutions present onindividual members of a population of proteins. In certain embodiments,such control results in a decrease in the amount of NGA2F+NGA2F-GlcNacoligosaccharides and/or an increase in the amount of NA1F+NA2Foligosaccharides linked to the protein of interest. In certainembodiments, such control results in an increase in the amount ofNGA2F+NGA2F-GlcNac oligosaccharides and/or a decrease in the amount ofNA1F+NA2F oligosaccharides linked to the protein of interest.

In certain embodiments, control over protein glycosylation heterogeneityis exerted by employing specific hydrolysates during production of theprotein of interest, for example, but not by way of limitation, inadaptation cultures performed in media supplemented with hydrolysates.In certain embodiments, control over protein glycosylation heterogeneityis exerted by maintaining certain yeastolate to phytone ratios duringproduction of the protein of interest. In certain embodiments, controlover protein glycosylation heterogeneity is exerted by the addition ofasparagine during the production of the protein of interest. In certainembodiments the amount of asparagine present in the cell culture mediawill range from about 0 mM to about 26 mM.

In certain embodiments, control over the heterogeneity of the proteincompositions described herein is exerted by employing one or more of theforegoing methods during the production and purification of the desiredproteins, such as antibodies or antigen-binding portions thereof,described herein.

The heterogeneity of the proteins of interest in the resultant sampleproduct can be analyzed using methods well known to those skilled in theart, e.g., weak cation exchange chromatography (WCX), capillaryisoelectric focusing (cIEF), size-exclusion chromatography, Poros™ AHPLC Assay, Host Cell Protein ELISA, Protein A ELISA, and western blotanalysis.

In yet another embodiment, the invention is directed to one or morepharmaceutical compositions comprising an isolated protein, such as anantibody or antigen-binding portion thereof, and an acceptable carrier.In another aspect, the compositions further comprise one or morepharmaceutically acceptable carriers, diluents, and/or pharmaceuticalagents.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict the effect of yeast, soy or wheat hydrolysateaddition to CDM GIA-1 in adalimumab-producing CHO cell line #1 on (FIG.1A) Culture growth, (FIG. 1B) Culture viability and (FIG. 1C) Harvesttiter.

FIGS. 2A and 2B depict the effect of yeast, soy or wheat hydrolysateaddition to CDM GIA-1 in adalimumab-producing CHO cell line #1 on (FIG.2A) NGA2F+NGA2F-GlcNac and (FIG. 2B) NA1F+NA2F.

FIGS. 3A-3C depict the effect of combined supplementation of yeast andsoy hydrolysates to CD media from multiple suppliers inadalimumab-producing CHO cell line #1 on (FIG. 3A) Culture growth, (FIG.3B) Culture viability and (FIG. 3C) Harvest titer.

FIGS. 4A and 4B depict the effect of combined supplementation of yeastand soy hydrolysates to CD media from multiple suppliers inadalimumab-producing CHO cell line #1 on (FIG. 4A) NGA2F+NGA2F-GlcNacand (FIG. 4B) NA1F+NA2F.

FIGS. 5A-5C depict the effect of supplementing (FIG. 5A) yeast, (FIG.5B) soy, or (FIG. 5C) wheat hydrolysate from multiple vendors to CDMGIA-1 on culture growth in CHO cell line #1.

FIGS. 6A-6C depict the effect of supplementing (FIG. 6A) yeast, (FIG.6B) soy, or (FIG. 6C) wheat hydrolysate from multiple vendors to CDMGIA-1 on culture viability in CHO cell line #1.

FIG. 7 depicts the effect of supplementing yeast, soy, or wheathydrolysate from multiple vendors to CDM GIA-1 on harvest titer in CHOcell line #1.

FIGS. 8A and 8B depict the effect of supplementing yeast, soy, or wheathydrolysate from multiple vendors to CDM GIA-1 in CHO cell line #1 on(FIG. 8A) NGA2F+NGA2F-GlcNac and (FIG. 8B) NA1F+NA2F.

FIG. 9A depicts viable cell density and FIG. 9B depicts viability inExample 4: Hydrolysate study #1 using distinct ratios of yeast to soyhydrolysate in adalimumab-producing CHO cell line #1.

FIG. 10A depicts viable cell density and FIG. 10B depicts viability inExample 4: Hydrolysate study #2 using distinct ratios of yeast to soyhydrolysate in adalimumab-producing CHO cell line #1.

FIG. 11 depicts the glycosylation profile in Example 4: HydrolysateStudy #1 in adalimumab-producing CHO cell line #1.

FIG. 12 depicts the glycosylation profile in Example 4: HydrolysateStudy #2 in adalimumab-producing CHO cell line #1.

FIGS. 13A-13C depict the effect of supplementation of asparagine and/orglutamine on day 6 to hydrolysate based media in CHO cell line #1 onculture growth (FIG. 13A), culture viability (FIG. 13B) and producttiter (FIG. 13C).

FIGS. 14A and 14B depict the effect of supplementation of asparagineand/or glutamine on Day 6 to hydrolysate based media inadalimumab-producing CHO cell line #1 on NGA2F and NGA2F-GlcNac glycans(FIG. 14A) and on NA1F and NA2F glycans (FIG. 14B).

FIGS. 15A-15C depict the dose dependent effect of supplementation ofasparagine on Day 7 to hydrolysate based media in adalimumab-producingCHO cell line #1 on culture growth (FIG. 15A), culture viability (FIG.15B) and product titer (FIG. 15C).

FIGS. 16A and 16B depict] the dose dependent effect of supplementationof asparagine on Day 7 to hydrolysate based media inadalimumab-producing CHO cell line #1 on NGA2F and NGA2F-GlcNac glycans(FIG. 16A) and on NA1F and NA2F glycans (FIG. 16B).

FIGS. 17A-17C depict the dose dependent effect of supplementation ofasparagine on Day 0 to hydrolysate based media in adalimumab-producingCHO cell line #1 on culture growth (FIG. 17A), culture viability (FIG.17B) and product titer (FIG. 17C).

FIGS. 18A and 18B depict the dose dependent effect of supplementation ofasparagine on Day 0 to hydrolysate based media in adalimumab-producingCHO cell line #1 on NGA2F and NGA2F-GlcNac glycans (FIG. 18A) and onNA1F and NA2F glycans (FIG. 18B).

FIGS. 19A-19C depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM Irvine IS CHO-CD in adalimumab-producing CHO cell line#1 on (FIG. 19A) Culture growth, (FIG. 1B) Culture viability and (FIG.19C) Harvest titer.

FIGS. 20A and 20B depict the effect of yeast, soy, or wheat hydrolysatesaddition to CDM Irvine IS CHO-CD in adalimumab-producing CHO cell line#1 on oligosaccharides profile (FIG. 20A) NGA2F+NGA2F-GlcNac and (FIG.20B) NA1F+NA2F.

FIGS. 21A-21C depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM GIA-1 in adalimumab-producing CHO cell line #2 on (FIG.21A) Culture growth, (FIG. 21B) Culture viability and (FIG. 21C) Harvesttiter.

FIGS. 22A and 22B depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM GIA-1 in adalimumab-producing CHO cell line #2 on (FIG.22A) NGA2F+NGA2F-GlcNac and (FIG. 22B) NA1F+NA2F.

FIGS. 23A-23C depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM GIA-1 in adalimumab-producing CHO cell line #3 on (FIG.23A) Culture growth, (FIG. 23B) Culture viability and (FIG. 23C) Harvesttiter.

FIGS. 24A and 24B depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM GIA-1 in adalimumab-producing CHO cell line #3 on (FIG.24A) NGA2F+NGA2F-GlcNac and (FIG. 24B) NA1F+NA2F.

FIGS. 25A-25C depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM GIA-1 in CHO cell line producing mAb #1 on (FIG. 25A)Culture growth, (FIG. 25B) Culture viability and (FIG. 25C) Harvesttiter.

FIGS. 26A and 26B depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM GIA-1 in CHO cell line producing mAb #1 on (FIG. 26A)NGA2F+NGA2F-GlcNac and (FIG. 26B) NA1F+NA2F.

FIGS. 27A-27C depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM GIA-1 in CHO cell line producing mAb #2 on (FIG. 27A)Culture growth, (FIG. 27B) Culture viability and (FIG. 27C) Harvesttiter.

FIGS. 28A and 28B depict the effect of yeast, soy, or wheat hydrolysateaddition to CDM GIA-1 in CHO cell line producing mAb #2 on (FIG. 28A)NGA2F+NGA2F-GlcNac and (FIG. 28B) NA1F+NA2F.

FIGS. 29A-29C depict the effect of combined supplementation of yeast,soy and/or wheat hydrolysates to CDM GIA-1 in adalimumab-producing CHOcell line #1 on (FIG. 29A) Culture growth, (FIG. 29B) Culture viabilityand (FIG. 29C) Harvest titer.

FIGS. 30A and 30B depict the effect of combined supplementation ofyeast, soy, and/or wheat hydrolysates to CDM GIA-1 inadalimumab-producing CHO cell line #1 on (FIG. 30A) NGA2F+NGA2F-GlcNacand (FIG. 30B) NA1F+NA2F.

FIGS. 31A-31C depict the dose dependent effect of supplementation ofasparagine on Day 6 to CDM GIA-1 in adalimumab-producing CHO cell line#1 on culture growth (FIG. 31A) and culture viability (FIG. 31B) andproduct titer (FIG. 31C).

FIGS. 32A and 32B depict the dose dependent effect of supplementation ofasparagine on Day 6 to CDM GIA-1 in adalimumab-producing CHO cell line#1 on NGA2F and NGA2F-GlcNac glycans (FIG. 32A) and on NA1F and NA2Fglycans (FIG. 32B).

FIGS. 33A-33C depict the dose dependent effect of supplementation ofasparagine on Day 6 to CDM GIA-1 in adalimumab-producing CHO cell line#2 on culture growth (FIG. 33A) and culture viability (FIG. 33B) andproduct titer (FIG. 33C).

FIGS. 34A and 34B depict the dose dependent effect of supplementation ofasparagine on Day 6 to CDM GIA-1 in adalimumab-producing CHO cell line#2 on NGA2F and NGA2F-GlcNac glycans (FIG. 34A) and on NA1F and NA2Fglycans (FIG. 34B).

FIGS. 35A-35C depict the dose dependent effect of supplementation ofasparagine during medium preparation to CDM GIA-1 in CHO cell lineproducing mAb #2 on culture growth (FIG. 35A) and culture viability(FIG. 35B) and product titer (FIG. 35C).

FIGS. 36A and 36B depict the dose dependent effect of supplementation ofasparagine during medium preparation to CDM GIA-1 in CHO cell lineproducing mAb #2 on NGA2F and NGA2F-GlcNac glycans (FIG. 36A) and onNA1F and NA2F glycans (FIG. 36B).

FIGS. 37A-37C depict the dose dependent effect of supplementation ofasparagine on Day 5 to CDM GIA-1 in CHO cell line producing mAb #2 onculture growth (FIG. 37A) and culture viability (FIG. 37B) and producttiter (FIG. 37C).

FIGS. 38A and 38B depict] the dose dependent effect of supplementationof asparagine on Day 5 to CDM GIA-1 in CHO cell line producing mAb #2 onNGA2F and NGA2F-GlcNac glycans (FIG. 38A) and on NA1F and NA2F glycans(FIG. 38B).

FIG. 39 depicts the experimental design for Example 1.

FIG. 40 depicts the experimental design for Example 2.

FIG. 41 depicts the experimental design for Example 3.

FIG. 42 depicts the experimental design for Example 6.

FIG. 43 depicts the experimental design for Example 7.

FIG. 44 depicts the experimental design for Example 8.

FIG. 45 depicts the experimental design for Example 9.

FIG. 46 depicts the experimental design for Example 10.

FIG. 47 depicts the experimental design for Example 11 (adaptationstage).

FIG. 48 depicts the experimental design for Example 11 (productionstage).

5. DETAILED DESCRIPTION

For clarity and not by way of limitation, this detailed description isdivided into the following sub-portions:

-   -   5.1 Definitions; and    -   5.2 Control of Heterogeneity:        -   5.2.1 Supplementation of CD Media with Yeast and/or Plant            Hydrolysates        -   5.2.2 Changing Yeast to Plant Hydrolysate Ratio in Cell            Culture Medium        -   5.2.3 Supplementation with Asparagine

5.1 Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

As used herein, the term “glycosylation” refers to the addition of acarbohydrate to an amino acid. Such addition commonly, although notexclusively, occurs via a nitrogen of asparagine or arginine (“N-linked”glycosylation) or to the hydroxy oxygen of serine, threonine, tyrosine,hydroxylysine, or hydroxyproline side-chains (“O-linked” glycosylation).In eukaryotes, N-linked glycosylation occurs on the asparagine of theconsensus sequence Asn-Xaa-Ser/Thr, in which Xaa is any amino acidexcept proline (Komfeld et al., Ann Rev Biochem 54: 631-664 (1985);Kukuruzinska et al, Proc. Natl. Acad. Sci. USA 84: 2145-2149 (1987);Herscovics et al, FASEB J. 7:540-550 (1993); and Orlean, SaccharomycesVol. 3 (1996)). O-linked glycosylation also takes place at serine orthreonine residues (Tanner et al., Biochim. Biophys. Acta. 906: 81-91(1987); and Hounsell et al, Glycoconj. J. 13: 19-26 (1996)). However,other glycosylation patterns can be formed, e.g., by linkingglycosylphosphatidyl-inositol to the carboxyl-terminal carboxyl group ofa protein.

The term “antibody” includes an immunoglobulin molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region (CH). The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., in the case of Adalimumab,hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment comprising the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentcomprising the VH and CH1 domains; (iv) a Fv fragment comprising the VLand VH domains of a single arm of an antibody, (v) a dAb fragment (Wardet al., (1989) Nature 341:544-546, the entire teaching of which isincorporated herein by reference), which comprises a VH domain; and (vi)an isolated complementarity determining region (CDR). Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv); see, e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883, the entire teachings of which are incorporated herein byreference). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (see,e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, theentire teachings of which are incorporated herein by reference). Stillfurther, an antibody or antigen-binding portion thereof may be part of alarger immunoadhesion molecule, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101, the entire teaching of which is incorporated herein byreference) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058,the entire teaching of which is incorporated herein by reference).Antibody portions, such as Fab and F(ab′)2 fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.In one aspect, the antigen binding portions are complete domains orpairs of complete domains.

As used herein, the term “recombinant host cell” (or simply “host cell”)refers to a cell into which a recombinant expression vector has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell but to the progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein. Incertain embodiments the host cell is employed in the context of a cellculture.

As used herein, the term “cell culture” refers to methods and techniquesemployed to generate and maintain a population of host cells capable ofproducing a recombinant protein of interest, as well as the methods andtechniques for optimizing the production and collection of the proteinof interest. For example, once an expression vector has beenincorporated into an appropriate host, the host can be maintained underconditions suitable for high level expression of the relevant nucleotidecoding sequences, and the collection and purification of the desiredrecombinant protein. Mammalian cells are preferred for expression andproduction of the recombinant of the present invention; however othereukaryotic cell types can also be employed in the context of the instantinvention. See, e.g., Winnacker, From Genes to Clones, VCH Publishers,N.Y., N.Y. (1987). Suitable mammalian host cells for expressingrecombinant proteins according to the invention include Chinese HamsterOvary (CHO cells) (including dhfr-CHO cells, described in Urlaub andChasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectablemarker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol.159:601-621, the entire teachings of which are incorporated herein byreference), NSO myeloma cells, COS cells and SP2 cells. Other,non-limiting, examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

When using the cell culture techniques of the instant invention, theprotein of interest can be produced intracellularly, in the periplasmicspace, or directly secreted into the medium. In embodiments where theprotein of interest is produced intracellularly, the particulate debris,either host cells or lysed cells (e.g., resulting from homogenization),can be removed by a variety of means, including but not limited to, bycentrifugation or ultrafiltration. Where the protein of interest issecreted into the medium, supernatants from such expression systems canbe first concentrated using a commercially available proteinconcentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit, which can then be subjected to one or moreadditional purification techniques, including but not limited toaffinity chromatography, including protein A affinity chromatography,ion exchange chromatography, such as anion or cation exchangechromatography, and hydrophobic interaction chromatography.

As used herein a “recombinant expression vector” can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host. For example, one of ordinary skill in the art wouldappreciate that transformation or transfection is a process by whichexogenous nucleic acid such as DNA is introduced into a cell wherein thetransformation or transfection process involves contacting the cell withthe exogenous nucleic acid such as the recombinant expression vector asdescribed herein. Non-limiting examples of such expression vectors arethe pUC series of vectors (Fermentas Life Sciences), the pBluescriptseries of vectors (Stratagene, LaJolla, Calif.), the pET series ofvectors (Novagen, Madison, Wis.), the pGEX series of vectors (PharmaciaBiotech, Uppsala, Sweden), and the pEX series vectors (Clontech, PaloAlto, Calif.).

As used herein, the term “recombinant protein” refers to a proteinproduced as the result of the transcription and translation of a genecarried on a recombinant expression vector that has been introduced intoa host cell. In certain embodiments the recombinant protein is anantibody, preferably a chimeric, humanized, or fully human antibody. Incertain embodiments the recombinant protein is an antibody of an isotypeselected from group consisting of: IgG (e.g., IgG1, IgG2, IgG3, IgG4),IgM, IgA1, IgA2, IgD, or IgE. In certain embodiments the antibodymolecule is a full-length antibody (e.g., an IgG1 or IgG4immunoglobulin) or alternatively the antibody can be a fragment (e.g., aFc fragment or a Fab fragment).

As used herein, the term “Adalimumab”, also known by its trade nameHumira® (Abbott Laboratories) refers to a human IgG antibody that bindsthe human form of tumor necrosis factor alpha. In general, the heavychain constant domain 2 (C_(H) 2) of the Adalimumab IgG-Fc region isglycosylated through covalent attachment of oligosaccharide atasparagine 297 (Asn-297). Adalimumab produced by Chinese hamster ovary(CHO) cells exists in 6 oligosaccharide forms, designated as NGA2F,NGA2F-GlcNAc, NA1F, NA2F, M5 and M6. Weak cation-exchange chromatography(WCX) analysis of the antibody has shown that it has three maincharged-variants (i.e. Lys 0, Lys 1, and Lys 2). These variants, or“charged isomers,” are the result of incomplete posttranslationalcleavage of the C-terminal lysine residues. In addition, WCX analysishas show that production of the antibody can result in the accumulationof two acidic species, identified herein as AR1 and AR2.

The term “about”, as used herein, is intended to refer to ranges ofapproximately 10-20% greater than or less than the referenced value. Incertain circumstances, one of skill in the art will recognize that, dueto the nature of the referenced value, the term “about” can mean more orless than a 10-20% deviation from that value.

The term “control”, as used herein, is intended to refer to bothlimitation as well as to modulation. For example, in certainembodiments, the instant invention provides methods for controllingdiversity that decrease the diversity of certain characteristics ofprotein populations, including, but not limited to, glycosylationpatterns. Such decreases in diversity can occur by: (1) promotion of adesired characteristic, such as a favored glycosylation pattern; (2)inhibition of an unwanted characteristic, such as a disfavoredglycosylation pattern; or (3) a combination of the foregoing. As usedherein, the term “control” also embraces contexts where heterogeneity ismodulated, i.e., shifted, from one diverse population to a secondpopulation of equal or even greater diversity, where the secondpopulation exhibits a distinct profile of the characteristic ofinterest. For example, in certain embodiments, the methods of theinstant invention can be used to modulate the types of oligosaccharidesubstitutions present on proteins from a first population ofsubstitutions to a second equally diverse, but distinct, population ofsubstitutions.

5.2 Control of Protein Heterogeneity

5.2.1 Supplementation of CD Media with Yeast and/or Plant Hydrolysates

It is well known that the pattern of glycoforms that arise inrecombinant proteins, including monoclonal antibodies, can be affectedby culture conditions during production. (Nam et al., The effects ofculture conditions on the glycosylation of secreted human placentalalkaline phosphatase produced in Chinese hamster ovary cells. BiotechnolBioeng. 2008 Aug. 15; 100(6): 1178-92). Consistency in the quality ofthe glycoproteins is important because glycosylation may impact proteinsolubility, activity, and circulatory half-life. (Gawlitzek et al.,Effect of Different Cell Culture Conditions on the Polypeptide Integrityand N-glycosylation of a Recombinant Model Glycoprotein. Biotechnol.Bioeng. 1995; 46:536-544; and Hayter et al., Glucose-limited ChemostatCulture of Chinese Hamster Ovary Cells Producing Recombinant HumanInterferon-γ. Biotechnol. Bioeng. 1992; 39:327-335).

In certain instances, such glycosylation-based heterogeneity can takethe form of differences in the galactose composition of N-linkedoligosaccharides. For example, a terminal galactose is added to NGA2F by(β-galactosyltransferase enzyme in the presence of manganese chloride,to produce NA1F (in the case of an addition of a single terminalgalactose) or NA2F (in the case of an addition of two terminal galactosemolecules). This galactosyltransferase-mediated reaction employsUDP-galactose as the sugar substrate and Mn²⁺ as a cofactor forgalactosyltransferase. Thus, without being bound by theory, it isbelieved that a change in protein homogeneity taking the form of anincrease in the fraction of N-linked oligosaccharide NGA2F and adecrease in the fraction of NA1F+NA2F N-linked oligosaccharides could becaused by either an insufficient amount of the substrate(UDP-galactose), the cofactor for galactosyltransferase (Mn²⁺), or both.

The experiments disclosed herein demonstrate that, in certainembodiments, supplementation of CD cell culture media with yeast and/orplant hydrolysates can modulate product quality of a mAb by, in certainembodiments, decreasing the NGA2F+NGA2F-GlcNac and, in certainembodiments, increasing the NA1F+NA2F oligosaccharides. These resultswere achieved in multiple CD media available from multiple vendors (LifeSciences Gibco, HyClone, and Irvine Scientific), using yeast and/orplant hydrolysates (for example, but not by way of limitation, soy,wheat, rice, cotton seed, pea, corn, and potato) from multiple vendors(BD Biosciences, Organotechnie, Sheffield/Kerry Biosciences, IrvineScientific, and DMV International). In experiments where yeast or planthydrolysates were added individually, a dose-dependent effect in theextent of reduction of NGA2F+NGA2F-GlcNac oligosaccharides (and acorresponding increase in the NA1F+NA2F oligosaccharides) withincreasing yeast or plant hydrolysates concentration in culture CD mediawas observed. For example, but not by way of limitation, yeasthydrolysates can be used to supplement a CD cell culture media atconcentrations ranging from about 2 g/L to about 11 g/L to achieve thedesired reduction of NGA2F+NGA2F-GlcNac oligosaccharides and acorresponding increase in the NA1F+NA2F oligosaccahrides. In certainnon-limiting embodiments, yeast hydrolysates can be used to supplement aCD cell culture media at concentrations of about 2 g/L, about 5 g/L, orabout 11 g/L. In certain non-limiting embodiments, plant hydrolysatescan be used to supplement a CD cell culture media at concentrationsranging from about 2 g/L to about 15 g/L to achieve the desiredreduction of NGA2F+NGA2F-GlcNac oligosaccahrides and a correspondingincrease in the NA1F+NA2F oligosaccharides. In certain non-limitingembodiments, plant hydrolysates can be used to supplement a CD cellculture media at concentrations of about 2 g/L, about 4 g/L, 7 g/L, 10g/L, or about 15 g/L.

In certain embodiments, the concentration of yeast and/or planthydrolysates is maintained in such a manner as to reduce theNGA2F+NGA2F-GlcNac sum in a protein or antibody sample by about 1%,1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, and ranges within one or more of the preceding. Incertain embodiments, the concentration of yeast and/or planthydrolysates is maintained in such a manner as to increase the NA1F+NA2Fsum in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%,2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, andranges within one or more of the preceding.

In certain embodiments, control over the glycosylation distribution ofproteins produced by cell culture can be exerted by maintaining theappropriate yeast hydrolysate concentration in the cell cultureexpressing the protein of interest as described herein. Specific cultureconditions can be used in various cultivation methods including, but notlimited to, batch, fed-batch, chemostat and perfusion, and with variouscell culture equipment including, but not limited to, shake flasks withor without suitable agitation, spinner flasks, stirred bioreactors,airlift bioreactors, membrane bioreactors, reactors with cells retainedon a solid support or immobilized/entrapped as in microporous beads, andany other configuration appropriate for optimal growth and productivityof the desired cell line

5.2.2 Changing Yeast to Plant Hydrolysate Ratio in Cell Culture Medium

The instant disclosure relates to control of the glycosylationdistribution in mammalian cell culture processes, including wherespecific components, such as hydrolyzed yeast and soy-based supplements,are commonly used and are typical constituents of suspension culturemedia in such processes. These nutrients are important for ensuring bothrobust cell growth and production of glycoproteins. However, the presentinvention utilizes these components in such a way to affect the criticalquality attributes of the glycoprotein. For example, but not by way oflimitation, by adjusting the concentration ratio of these twohydrolysates, yeast and soy (phytone), within the range of about 0.25 toabout 1.55, the resultant glycosylation distribution can be modified. Asoutlined in Example 1, non-limiting embodiments of the present inventioninclude supplements comprising 100% yeast hydrolysate as well as thosethat are 100% plant hydrolysate. Thus, this disclosure provides a meansto modulate glycosylation variations introduced by process inputs, suchas raw materials, and other variability inherent in dynamicmanufacturing operations. Ultimately, the disclosure enables in-processcontrol of protein glycosylation with respect to desired productspecifications.

In certain embodiments, the ratio of these two hydrolysates, yeast andsoy (phytone), is maintained in such a manner as to reduce theNGA2F+NGA2F-GlcNac sum in a protein or antibody sample by about 1%,1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, and ranges within one or more of the preceding. Incertain embodiments, the ratio of these two hydrolysates, yeast and soy(phytone), is maintained in such a manner as to increase the NA1F+NA2Fsum in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%,2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, andranges within one or more of the preceding.

In certain embodiments, control over the glycosylation distribution ofprotein produced by cell culture can be exerted by maintaining theappropriate yeast to plant hydrolysate ratio in the cell cultureexpressing the protein of interest as described herein. Specific cultureconditions can be used in various cultivation methods including, but notlimited to, batch, fed-batch, chemostat and perfusion, and with variouscell culture equipment including, but not limited to, shake flasks withor without suitable agitation, spinner flasks, stirred bioreactors,airlift bioreactors, membrane bioreactors, reactors with cells retainedon a solid support or immobilized/entrapped as in microporous beads, andany other configuration appropriate for optimal growth and productivityof the desired cell line

5.2.3 Supplementation with Asparagine

The instant disclosure relates to control of the glycosylationdistribution in mammalian cell culture processes, including wherespecific components, such as amino acids and amino acid-basedsupplements, are commonly used and are typical constituents ofsuspension culture media. These nutrients are important for ensuringboth robust cell growth and production of glycoproteins. However, thispresent invention utilizes these components, and in particularasparagine and/or glutamine in such a way to affect the critical qualityattributes of the glycoprotein. For example, but not by way oflimitation, by adjusting the concentration of one or both of these twoamino acids the resultant glycosylation distribution can be modified.Thus, this disclosure provides a means to modulate glycosylationvariations introduced by process inputs, such as raw materials, andother variability inherent in dynamic manufacturing operations.Ultimately, the disclosure enables in-process control of proteinglycosylation with respect to desired product specifications.

The experiments disclosed herein demonstrate that, in certainembodiments, supplementation of cell culture media with asparagineand/or glutamine can modulate product quality of a mAb by, in certainembodiments, increasing the NGA2F+NGA2F-GlcNac and, in certainembodiments, decreasing the NA1F+NA2F oligosaccharides. For example, butnot by way of limitation, the percentage of NGA2F+NGA2F-GlcNac can beincreased by 2-4% and the percentage of NA1F+NA2F was decreased by 2-5%when 0.4 to 1.6 g/L asparagine is added on either day 0 or days 6 or 7,as outlined in Example 5, below. Similarly, addition of 0.4 g/Lglutamine, to the culture run described in Example 5, below, increasedthe percentage of NGA2F+NGA2F-GlcNac by 1% and lowered the percentage ofNA1F+NA2F by 1%. Finally, adding both asparagine and glutamine (0.4 g/Lof each), to the cell culture run described in Example 5, below,increased the percentage of NGA2F+NGA2F-GlcNac by 3% and decreased thepercentage of NA1F+NA2F by 4%. In addition, the cell growth profile isthe same when 0.8 and 1.6 g/L of asparagine was added, but a dosedependent effect on oligosaccharide distribution was observed,indicating that the effect on oligosaccharide distribution was due tothe addition of asparagine and not the increased maximum viable celldensity or delayed drop in viability. In certain embodiments, the totalamount of asparagine in the cell culture media will range from about 0mM to about 26 mM. In certain embodiments, for example those embodimentswhere a hydrolysate media is employed, the range of asparagine in thecell culture media will range from about 1.3 mM to about 14.6 mM. Incertain embodiments, for example, but not limited to, those embodimentswhere GIA1 media is employed, the range of asparagine in the cellculture media will range from about 12.3 mM to about 25.7 mM.

In certain embodiments, the concentration of asparagine and/or glutamineis maintained in such a manner as to reduce the NA1F+NA2F sum in aprotein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%,3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and rangeswithin one or more of the preceding. In certain embodiments, theconcentration of asparagine and/or glutamine is maintained in such amanner as to increase the NGA2F+NGA2F-GlcNac sum in a protein orantibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%,4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one ormore of the preceding.

In certain embodiments, control over the glycosylation distribution ofprotein produced by cell culture can be exerted by maintaining theappropriate asparagine and/or glutamine concentration in the cellculture expressing the protein of interest as described herein. Specificculture conditions can be used in various cultivation methods including,but not limited to, batch, fed-batch, chemostat and perfusion, and withvarious cell culture equipment including, but not limited to, shakeflasks with or without suitable agitation, spinner flasks, stirredbioreactors, airlift bioreactors, membrane bioreactors, reactors withcells retained on a solid support or immobilized/entrapped as inmicroporous beads, and any other configuration appropriate for optimalgrowth and productivity of the desired cell line.

EXAMPLES Example 1 Control of Heterogeneity by Addition of Hydrolysatesto CD Media GIA-1 for Culture of an Adalimumab-Producing CHO Cell Line#1

Control of heterogeneity of therapeutic monoclonal antibodies (mAbs) canaid in ensuring their efficacy, stability, immunogenicity, andbiological activity. Media composition has been shown to play a role inproduct quality of mAbs together with process conditions and choice ofcell line. In certain embodiments, the present invention providesmethods for fine-tuning the product quality profile of a mAb produced invarious Chinese hamster ovary (CHO) cell lines by supplementation ofyeast and/or plant hydrolysates to chemically defined (CD) media. Incertain embodiments, the resulting mAb product is characterized byhaving a decreased content of complex agalactosylated glycans NGA2F andNGA2F-GlcNac and increased levels of terminally galactosylated glycansNA1F and NA2F. In certain embodiments, addition of increasing amounts ofyeast, soy or wheat hydrolysates from several suppliers to a CD mediumresulted in altered product quality profiles in aconcentration-dependent manner.

In the studies summarized in this example, the effects on glycosylationresulting from the addition of yeast (Bacto TC Yeastolate: 2, 5, 11g/L), soy (BBL Phytone Peptone: 2, 4, 7, 10, 15 g/L), or wheat (WheatPeptone E1: 2, 4, 7, 10, 15 g/L) hydrolysates to CD medium GIA-1 (LifeTechnologies Gibco; proprietary formulation) in the adalimumab-producingCHO cell line #1 were investigated.

1.1 Materials and methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone, or Wheat Peptone E1 according to theexperimental design in FIG. 39. The control cultures were notsupplemented with hydrolysates. In addition to hydrolysates, adaptationmedia was supplemented with 0.876 g/kg L-glutamine and 2.0 mL/kgmethotrexate solution, and production media was supplemented with 0.584g/L L-glutamine. The experiment was designed into two blocks. All mediapH was adjusted to approximately 7.1 using 6N hydrochloric acid/5Nsodium hydroxide. The media osmolality was adjusted to 290-300 mOsmol/kgwith sodium chloride.

The adalimumab-producing cultures were expanded for 3 passages (3 dayseach) in their respective adaptation media in a combination of 250 mL(50 mL or 100 mL working volume) and 500 mL (150 mL working volume)Corning vented non-baffled shake flasks and maintained on an orbitalshaker at 110 RPM in a 35° C., 5% CO₂ dry incubator. At each passage,cultures were inoculated at an initial viable cell density (VCD) ofapproximately 0.5×10⁶ cells/mL.

Production cultures were initiated in duplicate 500 mL Corning, vented,non-baffled shake flasks each containing 200 mL culture in dryincubators at 35° C., 5% CO₂, and 110 RPM. Initial VCD was approximately0.5×10⁶ cells/mL. A 1.25% (v/v) 40% glucose stock solution was fed whenthe media glucose concentration was less than 3 g/L.

For all studies described throughout this application, samples werecollected daily and measured for cell density and viability using aCedex cell counter. Retention samples for titer analysis (2×1.5 mL percondition) via Poros A method were collected daily after cultureviability fell below 90%. Samples were centrifuged at 12,000 RPM for 5min and the supernatant was stored at −80° C. until further analysis.The harvest procedure was performed by centrifugation of the culturesample at 3,000 RPM for 30 min followed by storage of the supernatant in125 mL PETG bottles at −80° C. until protein A purification,oligosaccharide, and WCX-10 analysis.

For the oligosaccharide assay, the oligosaccharides are released fromthe protein by enzymatic digestion with N-glycanase. Once the glycansare released, the free reducing end of each glycan is labeled byreductive amination with a fluorescent tag, 2-aminobenzamide (2-AB). Theresulting labeled glycans are separated by normal-phase HPLC (NP-HPLC)in acetonitrile: 50 mM ammonium formate, pH 4.4, and detected by afluorescence detector. Quantitation is based on the relative areapercent of detected sugars. The relative area percents of theagalactosyl fucosylated biantennary oligosaccharides(NGA2F+[NGA2F-GlcNac]) and the galactose-containing fucosylatedbiantennary oligosaccharides NA1F+NA2F are reported and discussed.

1.2 Culture Growth and Productivity

The majority of cultures grew to a similar peak VCD in the range of9-11×10⁶ cells/mL. Cultures supplemented with 11 g/L yeast hydrolysateBD TC yeastolate experienced slight inhibition of growth (FIG. 1A).Viability profiles were comparable to the control condition withcultures lasting 11 to 13 days (FIG. 1B). Increasing the yeasthydrolysate concentration in CDM media GIA-1 resulted in decreasedaverage productivity compared to the control condition. Culturessupplemented with soy or wheat hydrolysates lasted 12 to 13 days, andexperienced slightly increased average titer compared to the controlcondition (FIG. 1C).

1.3 Oligosaccharide Analysis

Addition of yeast, soy, or wheat hydrolysates to CD media GIA-1 loweredthe percentage of glycans NGA2F+NGA2F-GlcNac by 1-14% and increased thepercentage of NA1F+NA2F glycans by 2-21% compared to control condition(NGA2F+NGA2F-GlcNac: 89%; NA1F+NA2F: 6%) (FIGS. 2A-B). A dose-dependentdecrease in NGA2F+NGA2F-GlcNac and a corresponding increase in NA1F+NA2Fglycans was observed with the addition of yeast, soy, or wheathydrolysate over the tested range. The highest percentage decrease inNGA2F+NGA2F-GlcNac and corresponding highest increase in NA1F+NA2Fglycans was recorded for the condition supplemented with 7 g/L BD BBLphytone peptone (NGA2F+NGA2F-GlcNac: 78%, and NA1F+NA2F: 18%) comparedto control.

Example 2 Yeast and Soy Hydrolysates Combined Addition to MultipleCommercially Available CD Media for Culture of an Adalimumab-ProducingCHO Cell Line #1

In the study summarized in this example, the effects of combined yeastand soy hydrolysates addition to CD media from multiple suppliers: LifeTechnologies Gibco (OptiCHO and GIA-1), Irvine Scientific (IS CHO-CD),and HyClone/Thermo Scientific (CDM4CHO) on product quality in theadalimumab-producing CHO cell line #1 utilized in Example 1 wereevaluated.

2.1 Materials and Methods

The liquid or powder formulation media were purchased from multiplevendors (Life Technologies Gibco—OptiCHO and GIA-1; Irvine Scientific—ISCHO-CD; and HyClone/Thermo Scientific—CDM4CHO), reconstituted per themanufacturers' recommendations, and supplemented with Bacto TCYeastolate and BBL Phytone Peptone according to the experimental designin FIG. 40. The control cultures for each condition were notsupplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 using 6N hydrochloric acid/5N sodium hydroxide.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 110 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended batch-mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

2.2 Culture Growth and Productivity

Commercially available CD media supported markedly different culturegrowth profiles with maximum VCD of 2-9×10⁶ cells/mL and cultureduration ranging from 7 to 15 days (FIG. 3A). Addition of yeast and soyhydrolysates to Life Technologies Gibco OptiCHO and GIA-1, and HyCloneCDM4CHO media decreased peak VCD and increased culture length by 2 to 6days. However, addition of hydrolysates to Irvine IS CHO-CD mediaincreased peak VCD from 2.5×10⁶ cells/mL to 5.4×10⁶ cells/mL. Cultureviability declined slower with addition of hydrolysates for all mediatested (FIG. 3B). Productivity also varied significantly among cultures;however, the addition of hydrolysates to CD media increased productivityin all cases (FIG. 3C).

2.3 Oligosaccharide Analysis

The combined addition of yeast and soy hydrolysates to variouscommercially available CD media lowered the percentage ofNGA2F+NGA2F-GlcNac glycans by 2-10% compared to control (FIG. 4A): from81% to 79% (HyClone CDM4CHO); from 80% to 75% (Irvine IS CHO-CD); from88% to 80% (Life Technologies OptiCHO); from 90% to 80% (LifeTechnologies GIA-1). The percentage of NA1F+NA2F glycans increased by3-8% compared to control (FIG. 4B): from 15% to 18% (HyClone CDM4CHO);from 6% to 21% (Life Technologies GIA-1); from 16% to 21% (Irvine ISCHO-CD); from 5% to 13% (Life Technologies OptiCHO).

Example 3 Supplementation of Yeast, Soy and Wheat Hydrolysates fromMultiple Vendors to CD Media GIA-1 for Culture of anAdalimumab-Producing CHO Cell Line #1

In the study summarized in this example, we investigated the effects onglycosylation resulting from the addition of yeast (5, 11 g/L), soy (4,7 g/L) or wheat (4, 7 g/L) hydrolysates from multiple vendors (BDBiosciences, Sheffield/Kerry Biosciences, DMV International, IrvineScientific, and Organotechnie) to CDM GIA-1 in the adalimumab-producingCHO cell line #1.

3.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone, or Wheat Peptone E1 according to theexperimental design in FIG. 41. The control cultures were notsupplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 using 6N hydrochloric acid/5N sodium hydroxide. Themedia osmolality was adjusted to 290-300 mOsmol/kg with sodium chloride.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 110 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning, vented, non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

3.2 Culture Growth and Productivity

Culture growth and viability profiles were comparable among all testconditions (FIGS. 5A-C, 6A-C) except for 11 g/L BD Bacto TC yeastolate,for which a slight decrease in the growth rate and maximum VCD wasobserved. Supplementation of CD media GIA-1 with yeast hydrolysateslowered the harvest titer by up to 25% compared to the control, whilethe harvest titer increased up to 14% and 27% with the addition of soyor wheat hydrolysates, respectively (FIG. 7).

3.3 Oligosaccharide Analysis

Addition of yeast, soy or wheat hydrolysates to CD media GIA-1 decreasedthe NGA2F+NGA2F-GlcNac glycans in a dose-dependent manner for allhydrolysate vendors evaluated (FIGS. 8A-B). Addition of yeasthydrolysates to CD media GIA-1 lowered the percentage ofNGA2F+NGA2F-GlcNac glycans by 4-9%, and increased the percentage ofNA1F+NA2F glycans by 5-10% compared to control (NGA2F+NGA2F-GlcNac: 90%;NA1F+NA2F: 6%). Addition of soy hydrolysates to CD media GIA-1 decreasedthe NGA2F+NGA2F-GlcNac glycans by 9-14%, and increased the NA1F+NA2Fglycans by 11-15% compared to control. Addition of wheat hydrolysatesdecreased the NGA2F+NGA2F-GlcNac glycans by 4-11%, and increased theNA1F+NA2F glycans by 6-21% compared to control.

Example 4 Control of Heterogeneity by Addition of Reduced Ratio of Yeastto Plant Hydrolysate

To identify the role which the ratio of yeast to plant hydrolysate playsin connection with the generation of protein heterogeneity, experimentsemploying a range of different hydrolysate ratios were undertaken. Thecell culture medium employed in each experimental process contains bothyeast and soy hydrolysate (phytone). The ratios of yeast to soyhydrolysate (by weight) are 1.55, 0.67 and 0.25. The total weight ofyeastolate and soy hydrolysate were not changed in each experimentalprocess. Two distinct yeastolate lots were used in connection with theseexperiments (see FIGS. 9 & 11 and 10 & 12, respectively). Culturegrowth, productivity and product quality were assessed. As outlined inFIGS. 9-12, reducing the yeast to soy hydrolysate ratio resulted inaltered oligosaccharide profiles.

4.1. Materials and Methods

The CHO cell line #1 was employed in the studies covered here. Theproduction medium used in this experiment contains basal medium PFCHO,Bacto TC yeastolate and phytone peptone. The pH of all media wasadjusted to 7.15; and media osmolality was adjusted to 373-403 mOsmol/kgwith sodium chloride. For each experiment, 500 mL shakers with 200 mLworking volume were employed at the following conditions: 35° C.constant temperature; 5% CO_(2;) and 110 RPM. Cultures were inoculatedat an initial viable cell density (VCD) of approximately 0.5×10⁶cells/mL. Two mL of 40% w/w glucose solution was added to each shakerwhen the glucose concentration dropped below 2 g/L. The shakers wereharvested when the viable cell density decreased to approximately 50%.The harvest broth was centrifuged at 3200 rpm for 30 min at 5° C. toremove cells and the supernatant was stored at −80° C.

Samples were taken daily from each shaker to monitor growth. Thefollowing equipment was used to analyze the samples: Cedex cell counter,Radiometer blood gas analyzer, YSI glucose analyzer, and osmometer. Theharvest samples stored at −80° C. were later thawed and analyzed fortiter with Poros A HPLC method. In addition, the thawed samples werefiltered through a 0.2 μm filter, purified by Protein A chromatography,and then oligosaccharide analysis was performed as described in Example1.

4.2 Cell Growth and Productivity

In the first hydrolysate study, the viable cell densities for thereduced ratios of yeastolate to phytone (i.e. Y/P=0.67 and Y/P=0.25)were much lower than the viable cell density for the 1.55 ratio ofyeastolate to phytone (FIG. 9). As a result, the IVCC on day 13 (i.e.the harvest day) was significantly lower for the reduced ratioconditions compared to the 1.55 ratio condition, and the titer was alsolower (but not statistically significantly—data not shown). Theviability profiles were comparable until day 8 (FIG. 9). After day 8,the viability declined faster for the reduced ratio conditions. Inhydrolysate study 2, the viable cell density and viability for the 1.55ratio were slightly lower than those with reduced ratio in theexponential phase, but higher in the decline phase (FIG. 10). However,the titer for the 1.55 ratio shaker was 0.2 g/L lower than the reducedratio (i.e. Y/P=0.67) (data not shown).

4.3. Oligosaccharide Analysis

Glycosylation profiles for hydrolysate studies 1 and 2 are shown inFIGS. 11 and 12, respectively. Reducing the ratio of yeastolate tophytone reduced the percentage of NGA2F+(NGA2F-GlcNAc) glycan. Inhydrolysate study 1, the percentage of NGA2F+(NGA2F-GlcNAc) wassignificantly reduced for Y/P=0.67 and Y/P=0.25 as compared to Y/P=1.55.The p values were 0.03 and 0.001 for Y/P=0.67 and Y/P=0.25,respectively. At the same time, the percentage of NA1F+NA2F wasincreased significantly as the ratio of yeastolate to phytone wasreduced.

As shown in FIG. 12 in hydrolysate study 2, the difference in thepercentage of NGA2F+(NGA2F-GlcNAc) between Y/P=0.67 and Y/P=1.55 wassignificant (i.e. p=0.000002). The percentage of NGA2F+(NGA2F-GlcNAc)was lowered from 77.5% in the 1.55 ratio to approximately 75.4% with thereduced ratio.

Therefore, this study successfully demonstrated that reducing the ratioof yeastolate to phytone could alter oligosaccharide profile using twodifferent lots of yeast hydrolysate.

Example 5 Control of Heterogeneity by Supplementation with Asparagine

The present invention relates to methods for modulating theglycosylation profile of a monoclonal antibody (mAb) by varying theconcentration of asparagine in cell culture media. Cell culture mediumcomponents, such as asparagine, are commonly used and are typicalconstituents of suspension culture media. These nutrients are importantfor ensuring both robust cell growth and production of glycoproteins. Ithas been shown that the cell viability and product titer can be enhancedby the addition of asparagine to a glutamine-free production medium(Genentech, Inc. “Production of Proteins in Glutamine-Free Cell CultureMedia” WO2011019619 (2010)). However, the present invention providesmethods to modify glycosyaltion distribution by adjusting theconcentration of asparagine. Without being bound by theory, it isthought that the effect of asparagine on glycosylation profile of anantibody is through its conversion to glutamine and/or aspartate.Asparagine is the amide donor for glutamine and can be converted toglutamine and/or aspartate (H Huang, Y Yu, X Yi, Y Zhang “Nitrogenmetabolism of asparagine and glutamate in Vero cells studied by 1 H/15 NNMR spectroscopy” Applied microbiology and biotechnology 77 (2007)427-436). Glutamine and aspartate are important intermediates inpyrimidine synthesis; and it is known that enhancing de novo pyrimidinebiosynthesis could increase intracellular UTP concentration (Genentech,Inc. “Galacosylation of Recombinant Glycoproteins” US20030211573(2003)). In addition, studies have suggested that glutamine andaspartate limitation is expected to inhibit amino sugar synthesis (G BNyberg, R R Balcarcel, B D Follstad, G Stephanopoulos, D I Wang“Metabolic effects on recombinant interferon-gamma glycosylation incontinuous culture of Chinese hamster ovary cells” Biotechnology andBioengineering 62 (1999) 336-47; DCF Wong, KTK Wong, L T Goh, C K Heng,MGS. Yap “Impact of dynamic online fed-batch strategies on metabolism,productivity and N-glycosylation quality in CHO cell cultures”Biotechnology and Bioengineering 89 (2005) 164-177). Both UTP and aminosugar are required for the synthesis of UDP-GlcNac, which is thesubstrate for protein glycosylation process. It is also possible thatthe effect of asparagine on glycosyaltion is via increasing ammoniaconcentration in the cell culture medium since it is showed that theaddition of ammonia in CHO cultures could reduce the extent ofglycosylation of synthesized EPO (M. Yang and M. Butler “Effect ofAmmonia on the Glycosylation of Human Recombinant Erythropoietin inCulture” Biotechnol. Prog. 16 (2000) 751-759). We have found thatammonia concentration was increased after asparagine addition into thecell culture media.

In the studies summarized in Example 5, we investigated the effects onproduct quality attributes resulting from the addition of asparagine tohydrolysate based medium in an adalimumab-producing CHO cell line,generically named CHO cell line #1. Two experiments were performed inthe instant Example. For the first experiment, glutamine and/orasparagine were added (at an individual concentration of 0.4 g/L) on day6. For the second experiment, asparagine was added at different dosage(i.e. 0.4 g/L, 0.8 g/L or 1.6 g/L) either on day 0 (before inoculation)or together with the first glucose shot (happened on day 7).

5.1 Materials and Methods

The CHO cell line #1 was employed in the studies covered here. Uponthaw, cells were expanded in a 19-days seed train and then transferredinto seed reactors for up to 7 days in growth medium. The cells werethen brought to the laboratory and used in the small scale bioreactorexperiments. The media used in these experiments contains basal mediaPFCHO (proprietary formulation), Bacto TC Yeastolate and PhytonePeptone.

Three litter Applikon bioreactors were sterilized and then charged withproduction medium. At inoculation, cells were aseptically transferredinto each bioreactor to reach an initial cell density of 0.5×10⁶ viablecells/mL. After inoculation, the bioreactors were set to the followingconditions: pH=7.1, T=35° C., DO=30%, and agitation=200 rpm. The pH wasshifted from 7.1 to 6.9 over the first 2.5 days and held at 6.9 for theremainder of the run. The percentage of dissolved oxygen was controlledby sparging a mixture of air and oxygen. The addition of 0.5 N NaOH orsparging of CO₂ maintained the pH. When the glucose concentration fellbelow 2 g/L, approximately 1.25% (v/v) of glucose solution (400 g/kg)was added to the cell culture.

For the first experiment, glutamine and/or asparagine were added (at anindividual concentration of 0.4 g/L) together with the first glucoseshot (happened on day 6). For the second experiment, asparagine wasadded at different dosage (i.e. 0.4 g/L, 0.8 g/L or 1.6 g/L) either onday 0 (before inoculation) or together with the first glucose shot(happened on day 7).

Samples were taken daily from each reactor to monitor growth. Thefollowing equipment was used to analyze the samples: Cedex cell counterfor cell density and viability; Radiometer ABL 5 blood gas analyzer forpH, pCO2 and pO2; YSI 7100 analyzer for glucose and lactateconcentration. Some of the daily samples and the harvest samples werecentrifuged at 3,000 RPM for 30 min and then the supernatants werestored at −80° C. Later, the thawed harvest samples were filteredthrough a 0.2 μm filter, purified by Protein A chromatography, and thenoligosaccharide analysis was performed and then oligosaccharide analysiswas performed as described in Example 1.

5.2 Culture Growth and Productivity

In both of the experiments performed in 3L bioreactor in hydrolysatebased media with CHO cell line #1 described in the instant Example, theaddition of glutamine and/or asparagine together with a glucose shotincreased the maximum cell density (FIG. 13A and 15A, respectively). Theincrease in cell density is started two days after the addition in bothcases. Maximum viable cell density was consistent when 0.4 g/L ofglutamine or asparagine was added. Increasing the concentration ofasparagine to 0.8 g/L or adding both glutamine and asparagine at aconcentration of 0.4 g/L each further increased the maximum viable celldensity; however, adding asparagine at a higher concentration than 0.8g/L (e.g., 1.6 g/L) did not continue to increase the maximum viable celldensity. In contrast, when asparagine was added on day 0 (beforeinoculation), the maximum viable cell density increased in a dosedependent manner, with the maximum viable cell density being reachedwhen 1.6 g/L of asparagine was added on day 0 (FIG. 17A).

A drop in viability was delayed, as compared to control cultures, inboth experiments described in the instant Example for approximately 3days when glutamine and/or asparagine was added on day 6 or 7 (FIGS. 13Band 15B, respectively). However, the drop in viability accelerated onthe last day of the cultures. In contrast, although the drop inviability was delayed when asparagine was added on day 0, the effect ofdelaying viability decay was not as efficient as when the amino acidswere added later (e.g., on day 6 or day 7) as shown in FIG. 17B.

5.3 Oligosaccharide Analysis

The experiments described in the instant Example indicate thatoligosaccharide distribution is altered with the addition of asparagineand/or glutamine. The addition of asparagine increasedNGA2F+NGA2F-GlcNac in a dose dependent manner. Compared to control, thepercentage of NGA2F+NGA2F-GlcNac was increased by 1.0-3.9% and thepercentage of NA1F+NA2F was decreased by 1.1-4.3% when 0.4 to 1.6 g/Lasparagine was added on either day 0 or days 6 or 7 (FIGS. 14A-14B,16A-16B and 18A-18B). Addition of 0.4 g/L glutamine increased thepercentage of NGA2F+NGA2F-GlcNac by 0.7% and lowered the percentage ofNA1F+NA2F by 0.9%. Adding both asparagine and glutamine (0.4 g/L ofeach) increased the percentage of NGA2F+NGA2F-GlcNAc by 3.3% anddecreased the percentage of NA1F+NA2F by 4.2%. In addition, the cellgrowth profile is the same when 0.8 and 1.6 g/L of asparagine was addedon day 7 (FIG. 15A and 15B), but a dose dependent effect onoligosaccharide distribution was observed (FIGS. 16A and 16B),indicating that the effect on oligosaccharide distribution was due tothe addition of asparagine and not the increased maximum viable celldensity or delayed drop in viability.

Example 6 Yeast, Soy, or Wheat Hydrolysate Addition to CommerciallyAvailable CD Media IS CHO-CD for Culture of an Adalimumab-Producing CHOCell Line #1

In the study summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia IS CHO-CD (Irvine Scientific) in the adalimumab-producing CHO cellline #1 utilized in Example 1 were evaluated.

6.1 Materials and methods

Adaptation and production media (Irvine Scientific IS CHO-CD 91119) weresupplemented with Bacto TC Yeastolate, BBL Phytone Peptone, or WheatPeptone E1 according to the experimental design in FIG. 42. The controlcultures were not supplemented with hydrolysates. All media pH wasadjusted to approximately 7.1.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 110 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

6.2 Culture Growth and Productivity

Addition of yeast, soy or wheat hydrolysates to Irvine IS CHO-CD mediaincreased the maximum VCD and culture length for most conditions studiedcompared to the control (FIG. 19A). The largest increase in maximum VCDwas recorded for cultures supplemented with 5 g/L Bacto TC Yeastolate. Aconcentration-dependent increase in harvest titer was observed for allcultures supplemented with hydrolysates (FIG. 19C).

6.3 Oligosaccharide Analysis

Supplementation of Irvine IS CHO-CD media with yeast hydrolysatesdecreased the percentage of NGA2F+NGA2F-GlcNac glycans by 3-4%, andincreased the percentage of NA1F+NA2F glycans by the same percentagecompared to control (NGA2F+NGA2F-GlcNac: 73%; NA1F+NA2F: 25%) (FIGS.20A-B). Addition of soy hydrolysates to Irvine IS CHO-CD media decreasedthe percentage of NGA2F+NGA2F-GlcNac glycans by 4%, and increased thepercentage of NA1F+NA2F glycans by the same percentage compared tocontrol. However, addition of wheat hydrolysates to Irvine IS CHO-CDmedia resulted in an opposite trend. A concentration-dependent increasein the percentage of NGA2F+NGA2F-GlcNac glycans by 1-3% and acorresponding decrease in the percentage of NA1F+NA2F glycans wasobserved.

Example 7 Yeast, Soy, or Wheat Hydrolysate Addition to CD Media GIA-1for Culture of an Adalimumab-Producing CHO Cell Line #2

In the study summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia GIA-1 in an adalimumab-producing CHO cell line, generically namedCHO cell line #2 were evaluated.

7.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone, or Wheat Peptone E1 according to theexperimental design in FIG. 43. The control cultures were notsupplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 and the media osmolality was adjusted to 290-300mOsmol/kg.

Cultures were expanded for 3 passages(3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 180 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

7.2 Culture Growth and Productivity

Supplementation of yeast, soy or wheat hydrolysates to CD media GIA-1extended the culture length by 1 to 3 days and decreased the maximum VCDin a dose-dependent manner (FIGS. 21A-B). The addition of thesehydrolysates at the highest concentrations significantly decreasedmaximum VCD, with wheat hydrolysates added at 10 g/L showing the mostsevere growth inhibition effects. However, an impact on harvest titerwas only observed for the culture supplemented with 10 g/L wheathydrolysates (65% reduction). An increase in the harvest titer comparedto the control (FIG. 21C) was found in most other cultures.

7.3 Oligosaccharide Analysis

Addition of yeast hydrolysates decreased the percentage ofNGA2F+NGA2F-GlcNac glycans by 3-5%, and increased the percentage ofNA1F+NA2F glycans by 5-8% compared to control (NGA2F+NGA2F-GlcNac: 89%;NA1F+NA2F: 3%) (FIGS. 22A-B). Addition of soy hydrolysates to CD mediaGIA-1 decreased the NGA2F+NGA2F-GlcNac glycans by 8-21%, and increasedthe NA1F+NA2F glycans by 10-15% compared to control. Addition of wheathydrolysates decreased the NGA2F+NGA2F-GlcNac glycans by 6-7%, andincreased the NA1F+NA2F glycans by 9-10% compared to control.

Example 8 Yeast, Soy, or Wheat Hydrolysate Addition to CD Media GIA-1for Culture of an Adalimumab-Producing CHO Cell Line #3

In the study summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia GIA-1 in an adalimumab-producing CHO cell line, generically namedCHO cell line #3 were evaluated.

8.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone, or Wheat Peptone E1 according to theexperimental design in FIG. 44. The control cultures were notsupplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 and the media osmolality was adjusted to 290-300mOsmol/kg.

Cultures were expanded for 3 passages(3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 140 RPM in a 36° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

8.2 Culture Growth and Productivity

Supplementation of production CD media with high concentrations ofhydrolysates—11 g/L yeast, 15 g/L soy or 15 g/L wheat hydrolysates,decreased the culture growth rate and increased the culture lengthcompared to the control (FIGS. 23A-B). Harvest titer increased withincreasing hydrolysate concentrations in the production media, exceptfor the condition supplemented with 15 g/L wheat hydrolysates, whichexperienced significant growth inhibition and harvest titer decreasecompared to control (FIG. 23C).

8.3 Oligosaccharide Analysis

Supplementation of CD media GIA-1 with yeast, soy or wheat hydrolysatesdecreased the percentage of NGA2F+NGA2F-GlcNac glycans and increased thepercentage of NA1F+NA2F glycans in a dose-dependent manner (FIGS.24A-B). Addition of yeast hydrolysates decreased the percentage ofNGA2F+NGA2F-GlcNac glycans by 5-21%, and increased the percentage ofNA1F+NA2F glycans by 3-11% compared to control (NGA2F+NGA2F-GlcNac: 91%;NA1F+NA2F: 6%). Addition of soy hydrolysates to CD media GIA-1 decreasedthe NGA2F+NGA2F-GlcNac glycans by 13-25%, and increased the NA1F+NA2Fglycans by 13-25% compared to control. Addition of wheat hydrolysatesdecreased the NGA2F+NGA2F-GlcNac glycans by 12-18%, and increased theNA1F+NA2F glycans by 12-18% compared to control.

Example 9 Yeast, Soy, or Wheat Hydrolysate Addition to CD Media GIA-1for Culture of a CHO Cell Line Producing mAb #1

In the studies summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia GIA-1 in a CHO cell line producing mAb #1 were evaluated.

9.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate (BD Biosciences; catalog #255772), BBL Phytone Peptone (BDBiosciences; catalog #211096), or Wheat Peptone E1 (Organotechnie;catalog #19559) according to the experimental design in FIG. 45. Thecontrol cultures were not supplemented with hydrolysates. All media pHwas adjusted to approximately 7.2 and the media osmolality was adjustedto 290-330 mOsmol/kg.

Cultures were expanded for 4 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an Infors Multitron orbital shaker at 140RPM in a 36° C., 5% CO₂ incubator. Production cultures were initiated induplicate 500 mL (200 mL working volume) Corning vented non-baffledshake flasks at approximately 1.0×10⁶ cells/mL initial VCD. The studywas run in an extended-batch mode by feeding a glucose solution (1.0%(v/v) of 40% solution) when the media glucose concentration fell below 3g/L.

9.2 Culture Growth and Productivity

Supplementation of yeast, soy or wheat hydrolysates to the CD mediaGIA-1 did not affect culture growth profiles dramatically (FIGS. 25A-B).There was some dose-dependent reduction of the peak VCD compared tocontrol as the hydrolysate concentrations increased, particularly in thecase of soy hydrolysates, but overall the growth profiles were similar.However, the culture duration was extended to 11-14 days compared to 9days for control. Cultures supplemented with 11 g/L yeast hydrolysatehad a substantial increase in harvest titer (FIG. 25C) that far exceededthe other conditions.

9.3 Oligosaccharide Analysis

Addition of yeast hydrolysates to CD media GIA-1 lowered the percentageof NGA2F+NGA2F-GlcNac glycans by 3%, and increased the percentage ofNA1F+NA2F glycans by 4% compared to control (NGA2F+NGA2F-GlcNac: 92%;NA1F+NA2F: 5%) (FIGS. 26A-B). Addition of soy hydrolysates lowered thepercentage of NGA2F+NGA2F-GlcNac glycans by 7-13%, and increased thepercentage of NA1F+NA2F glycans by 8-21% compared to control. Additionof wheat hydrolysates lowered the percentage of NGA2F+NGA2F-GlcNacglycans by 5-8%, and increased the percentage of NA1F+NA2F glycans by6-9% compared to control.

Example 10 Yeast, Soy, or Wheat Hydrolysate Addition to CD Media GIA-1for Culture of a CHO Cell Line Producing mAb #2

In the study summarized in this example, the effects on glycosylationresulting from the addition of yeast, soy or wheat hydrolysates to CDmedia GIA-1 in a CHO cell line producing mAb #2 were evaluated.

10.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TC, BBLPhytone Peptone, or Wheat Peptone E1 according to the experimentaldesign in FIG. 46. The control cultures were not supplemented withhydrolysates. All media pH was adjusted to approximately 7.2 and themedia osmolality was adjusted to 280-330 mOsmol/kg.

Upon thaw, cells were cultured in CD media GIA-1 growth media in acombination of Corning vented non-baffled shake flasks and maintained ona shaker platform at 140 RPM and 20 L cell bags. Cultures werepropagated in a 35° C., 5% CO₂ dry incubator. Production cultures wereinitiated in duplicate 500 mL (200 mL working volume) Corning ventednon-baffled shake flasks at an initial VCD of approximately 0.5×10⁶cells/mL. The shake flask study was run in an extended-batch mode byfeeding a glucose solution (1.25% (v/v) of 40% solution) when the mediaglucose concentration fell below 3 g/L. For this study, samples werecollected daily and measured for cell density and viability using a NOVAcell counter.

10.2 Culture Growth and Productivity

Supplementation of yeast, soy or wheat hydrolysates to CD media GIA-1did not impact culture growth for most conditions studied compared tocontrol (FIG. 27A). Supplementation with hydrolysates led to higherviability profiles compared to control (FIG. 27B). The addition of wheathydrolysates increased harvest titer compared to the control (FIG. 27C).

10.3 Oligosaccharide Analysis

Addition of yeast hydrolysates to CD media GIA-1 lowered the percentageof NGA2F+NGA2F-GlcNac glycans by 3% (FIG. 28A), and increased thepercentage of NA1F+NA2F glycans by 7% (FIG. 28B) in a dose-dependentmanner compared to control (NGA2F+NGA2F-GlcNac: 75%; NA1F+NA2F: 8%).Addition of soy hydrolysates lowered the percentage ofNGA2F+NGA2F-GlcNac by 2-21%, and increased the percentage of NA1F+NA2Fby 4-16% compared to control (NGA2F+NGA2F-GlcNac: 76%; NA1F+NA2F: 11%).For this cell line, there was no significant difference in thepercentage of NGA2F+NGA2F-GlcNac glycans between the control conditionand the cultures supplemented with wheat hydrolysates at theconcentration range evaluated. Furthermore, only a minor increase in thepercentage of NA1F+NA2F glycans was observed.

Example 11 Combined Yeast, Soy, and/or Wheat Hydrolysate Addition to CDMedia GIA-1 for Culture of an Adalimumab-Producing CHO Cell Line #1

In the study summarized in this example, the effects on glycosylationresulting from the individual or combined addition of yeast, soy, and/orwheat hydrolysates to CD media GIA-1 in the adalimumab-producing CHOcell line #1 utilized in Example 1 were evaluated.

11.1 Materials and Methods

Adaptation and production media were supplemented with Bacto TCYeastolate, BBL Phytone Peptone, and/or Wheat Peptone E1 according tothe experimental design in FIGS. 47 and 48. The control cultures werenot supplemented with hydrolysates. All media pH was adjusted toapproximately 7.1 and the media osmolality was adjusted to 290-300mOsmol/kg.

Cultures were expanded for 3 passages (3 days each) in their respectiveadaptation media in a combination of 250 mL (50 mL or 100 mL workingvolume) and 500 mL (150 mL working volume) Corning vented non-baffledshake flasks and maintained on an orbital shaker at 110 RPM in a 35° C.,5% CO₂ dry incubator. Production cultures were initiated in duplicate500 mL (200 mL working volume) Corning vented non-baffled shake flasksat an initial VCD of approximately 0.5×10⁶ cells/mL. The shake flaskstudy was run in an extended-batch mode by feeding a glucose solution(1.25% (v/v) of 40% solution) when the media glucose concentration fellbelow 3 g/L.

11.2 Culture Growth and Productivity

Supplementation of yeast, soy, and/or wheat hydrolysates to CD mediaGIA-1 resulted in slight growth inhibition and reduced maximum VCDcompared to the control (FIG. 29A). Culture viability profiles andharvest titer were comparable for all cultures (FIGS. 29B-C).

11.3 Oligosaccharide Analysis

Supplementation of yeast hydrolysates only to CD media GIA-1 decreasedthe percentage of NGA2F+NGA2F-GlcNac glycans by 4% and increased thepercentage of NA1F+NA2F glycans by 6% compared to control(NGA2F+NGA2F-GlcNac: 90%; NA1F+NA2F: 4%) (FIGS. 30A-B). Supplementationof soy hydrolysates only decreased the percentage of NGA2F+NGA2F-GlcNacglycans by 7%, and increased the percentage of NA1F+NA2F glycans by 9%compared to control. Supplementation of wheat hydrolysates onlydecreased the percentage of NGA2F+NGA2F-GlcNac glycans by 5% andincreased the percentage of NA1F+NA2F glycans by 8% compared to control.

The addition of two hydrolysates (yeast and soy; yeast and wheat; soyand wheat) further decreased the percentage of NGA2F+NGA2F-GlcNacglycans and increased the percentage of NA1F+NA2F glycans by a couple ofpercentages compared to the addition of each component individually(FIGS. 30A-B). Supplementing CD media GIA-1 with all three hydrolysatesdid not result in any further changes in the glycosylation profile,indicating a saturation state being reached.

Example 12 Effect of Asparagine in CD Media GIA-1 for Culture ofAdalimumab-Producing CHO Cell Line #1

In the study summarized in this Example, the effects on product qualityattributes resulting from the addition of asparagine to CD media GIA-1in an adalimumab-producing CHO cell line, generically named CHO cellline #1 were investigated.

12.1 Materials and Methods

The CHO cell line #1 was employed in the study covered here. Upon thaw,cells were expanded in a 19-days seed train and then transferred intoseed reactors for up to 7 days in growth medium. The cells were thenbrought to the laboratory and adapted in 500-mL shaker flasks with 200mL working volume in CD media GIA1 medium for 13 days with 3 passages.The shaker flasks were placed on a shaker platform at 110 RPM in a 35°C., 5% CO₂ incubator.

The chemical defined growth or production media, was prepared from basalIVGN CD media GIA1. For preparation of the IVGN CD media formulation,the proprietary media was supplemented with L-glutamine, insulin, sodiumbicarbonate, sodium chloride, and methotrexate solution. Productionmedia consisted of all the components in the growth medium, excludingmethotrexate. In addition, 5 mM of Galactose (Sigma, G5388) and 1004 ofManganese (Sigma, M1787) were supplemented into production medium.Osmolality was adjusted by the concentration of sodium chloride. Allmedia was filtered through filter systems (0.22 μm PES) and stored at 4°C. until usage.

Production cultures were initiated in duplicate 500 mL Corning, vented,non-baffled shaker flasks each containing 200 mL culture in dryincubators with 5% CO₂ at 35° C. and 110 RPM. Initial VCD wasapproximately 0.5×10⁶ cells/ml. The shake flask study was run in anextended batch mode by feeding a glucose solution (1.25% (v/v) of 40%solution) when the media glucose concentration fell below 3 g/L.Asparagine stock solution (20 g/L) was fed to culture on Day 6 toincrease Asparagine concentration by 0, 0.4, 1.2 and 2.0 g/L.

Samples were taken daily from each reactor to monitor growth. Thefollowing equipment was used to analyze the samples: Cedex cell counterfor cell density and viability; YSI 7100 analyzer for glucose andlactate concentration.

Some of the daily samples and the harvest samples were centrifuged at3,000rpm for 30 min and then supernatants were stored at −80° C. Thethawed harvest samples were subsequently filtered through a 0.2 μmfilter, purified by Protein A chromatography, and then oligosaccharideanalysis was performed as described in Example 1.

12.2 Culture Growth and Productivity

Feeding of asparagine to CD media GIA-1 did not impact culture growthfor most conditions studied as compared to the control (FIG. 31A). Thecultures showed similar growth rates and reached maximum VCD of ˜12×10⁶cells/mL. Culture viabilities were also very similar to that of thecontrols (FIG. 31B) Similarly, all the cultures examined here resultedin comparable harvest titers of approximately 1.7 g/L (FIG. 31C).

12.3 Oligosaccharide Analysis

The effect of asparagine addition on oligosaccharide distribution wasconsistent with the experiments performed in hydrolysate based mediadescribed above. The addition of asparagine increased NGA2F+NGA2F-GlcNAcglycans in a dose dependent manner (FIG. 32A). The percentage ofNGA2F+NGA2F-GlcNac in the control sample (without Asparagine addition)was as low as 74.7%. In the sample with the addition of asparagine thepercentage of NGA2F+NGA2F-GlcNAc was increased to 76.1% (0.4 g/L ofasparagine), 79.2% (1.2 g/L of asparagine), and 79.0% (2.0 g/L ofasparagine), for a total increase of 4.5%.

The percentage of NA1F+NA2F in the control sample (without asparagineaddition) was as high as 22.3% (FIG. 32B). In the sample with theaddition of asparagine the percentage of NA1F+NA2F was decreased to21.1% (0.4 g/L of asparagine), 17.8% (1.2 g/L of asparagine), and 17.8%(2.0 g/L of asparagine), for a total reduction of 4.5%.

Example 13 Effect of Asparagine in CD Media GIA-1 for Culture ofAdalimumab-Producing CHO Cell Line #3

In the study summarized in Example 13, the effects on product qualityattributes resulting from the addition of asparagine to CD media GIA-1in an adalimumab-producing CHO cell line, generically named CHO cellline #3 were investigated.

13.1 Materials and Methods

The CHO cell line #3 was employed in the study covered here. Upon thaw,adalimumab producing cell line #3 was cultured in CD media GIA-1 in acombination of vented shake flasks on a shaker platform @140 rpm and 20L wave bags. Cultures were propagated in a 36° C., 5% CO₂ incubator toobtain the required number of cells to be able to initiate productionstage cultures.

The chemical defined growth or production media was prepared from basalIVGN CD media GIA1. For preparation of the IVGN CD media formulation,the proprietary media was supplemented with L-glutamine, sodiumbicarbonate, sodium chloride, and methotrexate solution. Productionmedia consisted of all the components in the growth medium, excludingmethotrexate. In addition, 10 mM of Galactose (Sigma, G5388) and 0.204of Manganese (Sigma, M1787) were supplemented into production medium.Osmolality was adjusted by the concentration of sodium chloride. Allmedia was filtered through filter systems (0.22 μm PES) and stored at 4°C. until usage.

Production cultures were initiated in duplicate 500 mL Corning, vented,non-baffled shaker flasks each containing 200 mL culture in dryincubators with 5% CO₂ at 36° C. and 140 RPM. Initial VCD wasapproximately 0.5×10⁶ cells/ml. The shake flask study was run in anextended batch mode by feeding a glucose solution (1.25% (v/v) of 40%solution) when the media glucose concentration fell below 3 g/L.Asparagine stock solution (20 g/L) was fed to culture on Day 6 toincrease asparagine concentration by 0, 0.4, 0.8, 1.2, 1.6, and 2.0 g/L.

Samples were taken daily from each reactor to monitor growth. Thefollowing equipment was used to analyze the samples: Cedex cell counterfor cell density and viability; YSI 7100 analyzer for glucose andlactate concentration.

Some of the daily samples and the harvest samples were centrifuged at3,000 rpm for 30 min and then supernatants were stored at −80° C. Thethawed harvest samples were subsequently filtered through a 0.2 μmfilter, purified by Protein A chromatography, and then oligosaccharideanalysis was performed as described in Example 1.

13.2 Culture Growth and Productivity

The experiment described in the instant Example used a different cellline (i.e., CHO cell line #3) in CD media GIA-1. Culture growth andviability profiles were comparable among all test conditions withdifferent dosage of asparagine added on day 6 (FIGS. 33A and 33B). Allcultures reached maximum VCD of ˜18-19×10⁶ cells/mL. The product titer(˜1.5-1.6 g/L) was slightly reduced when higher dosage of asparagine wasadded (FIG. 33C).

13.3 Oligosaccharide Analysis

Again, the addition of asparagine increased NGA2F+NGA2F-GlcNac (FIG.34A). The percentage of NGA2F+NGA2F-GlcNac in the control sample(without asparagine addition) was as low as 68.7%. In the sample withthe addition of asparagine, the percentage of NGA2F+NGA2F-GlcNac wasincreased by 4.1-5.1% when 0.4 to 2.0 g/L asparagine was added on day 6(FIG. 34A). The percentage of NA1F+NA2F in the control sample (withoutasparagine addition) was as high as 25.6% (FIG. 34B). In the sample withthe addition of asparagine the percentage of NA1F+NA2F was decreased by3.8-4.6% when 0.4 to 2.0 g/L asparagine was added on day 6 (FIG. 34B).

Example 14 Effect of Asparagine in a Shaker Flask Batch Culture in CDMedia GIA-1 with a CHO Cell LineProducing mAb #2

In the studies summarized in Example 14, the effects on product qualityattributes resulting from the addition of asparagine to CD media GIA-1from Life Technologies Gibco in a CHO cell line producing monoclonalantibody #2 were investigated. In this instant Example, asparagine waseither supplemented into culture media during media preparation or addedon day 5 of the cell culture process.

14.1 Materials and Methods

mAb #2 producing cell line was employed in the study covered here. Uponthaw, cells were cultured in chemically defined growth media in acombination of vented baffled shake flasks (Corning) on a shakerplatform at 140 RPM. All media pH was adjusted to approximately 7.2 andthe media osmolality was adjusted to 280-330 mOsmol/kg.

Cultures were propagated in a 35° C., 5% CO₂ incubator to obtain therequired number of cells to be able to initiate production stagecultures. Production cultures were initiated in duplicate 500 mL ventednon-baffled Corning shake flasks (200 mL working volume) at an initialviable cell density (VCD) of approximately 0.5×10⁶ cells/mL. The shakeflask study was run in an extended batch mode by feeding a glucosesolution (1.25% (v/v) of 40% solution) when the media glucoseconcentration fell below 3 g/L. Asparagine (Sigma, Catalog Number A4284)were solubilized in Milli-Q water to make a 30 g/L stock solution. Allmedia was filtered through Corning or Millipore 1 L filter systems (0.22μm PES) and stored at 4° C. until usage.

For asparagine supplemented into culture media during media preparation,asparagine stock solution was supplemented to production media toincrease asparagine concentration by 0, 0.4, 0.8 and 1.6 g/L. Afteraddition of asparagine, media was brought to a pH similar tonon-supplemented (control) media using 5N hydrochloric acid/5N NaOH, andit was brought to an osmolality similar to non-supplemented (control)media by adjusting the concentration of sodium chloride. For asparagineaddition study, asparagine stock solution was added to culture on Day 5to increase Asparagine concentration by 0, 0.4, 0.8 and 1.6 g/L.

For all studies described throughout this invention, samples werecollected daily and measured for cell density and viability using a NOVAcell counter. Retention samples for titer analysis via Poros A methodwere collected by centrifugation at 12,000 RPM for 5 min when theculture viability began declining. The cultures were harvested bycollecting 125 mL aliquots and centrifuging at 3,000 RPM for 30 min whenculture viability was near or below 50%. All supernatants were stored at−80° C. until analysis. The harvest samples were Protein A purified andthen oligosaccharide analysis was performed as described in Example 1.

14.2 Culture Growth and Productivity

Adding asparagine to CD media GIA-1 during medium preparation or on day5 of the cell culture did not impact culture growth for most conditionsstudied as compared to the non-supplemented 0 g/L controls (FIGS. 45Aand 47A). The cultures showed similar growth rates and reached maximumVCD of 22-24×10⁶ cells/mL. Culture viabilities were also very similar tothat of the controls (FIGS. 35B and 37B). Similarly, all the culturesexamined here resulted in comparable harvest titers of approximately 0.9g/L of mAb #2 (FIGS. 35C and 37C).

14.3 Oligosaccharide Analysis

The addition of asparagine during medium preparation increasedNGA2F+NGA2F-GlcNac glycans in a dose dependent manner (FIG. 36A). Thepercentage of NGA2F+NGA2F-GlcNac in the control sample (withoutasparagine addition) was as low as 76.3%. In the sample with theaddition of asparagine the percentage of NGA2F+NGA2F-GlcNac wasincreased to 81.5% (0.4 g/L of asparagine), 85.5% (0.8 g/L ofasparagine), and 85.9% (1.6 g/L of asparagine), for a total increase of9.6%. The percentage of NA1F+NA2F in the control sample (withoutasparagine addition) was as high as 11.5% (FIG. 36B). In the sample withthe addition of asparagine the percentage of NA1F+NA2F was decreased to9.8% (0.4 g/L of asparagine), 7.8% (0.8 g/L of asparagine), and 7.0%(1.6 g/L of asparagine), for a total reduction of 4.5%. With mAb #2 cellline used in the study, the percentage of Mannose type glycans was alsodecreased with the supplementation of asparagine. The percentage ofMannoses in the control sample (without asparagine addition) was as highas 12.2% (FIG. 36B). In the sample with the addition of asparagine thepercentage of Mannoses was decreased to 8.6% (0.4 g/L of asparagine),6.7% (0.8 g/L of asparagine), and 7.1% (1.6 g/L of asparagine), for atotal reduction of 5.5%.

The addition of asparagine on day 5 of the culture also increasedNGA2F+NGA2F-GlcNac glycans in a dose dependent manner (FIG. 38A). Thepercentage of NGA2F+NGA2F-GlcNac in the control sample (withoutasparagine addition) was as low as 79.7%. In the sample with theaddition of asparagine the percentage of NGA2F+NGA2F-GlcNac wasincreased to 80.5% (0.4 g/L of asparagine), 82.1% (0.8 g/L ofasparagine), and 84.1% (1.6 g/L of asparagine), for a total increase of4.4%. The percentage of NA1F+NA2F in the control sample (withoutasparagine addition) was as high as 9.7% (FIG. 38B). In the sample withthe addition of asparagine the percentage of NA1F+NA2F was decreased to9.4% (0.4 g/L of asparagine), 9.6% (0.8 g/L of asparagine), and 8.5%(1.6 g/L of asparagine), for a total reduction of 1.2%. Again, thepercentage of Mannose type glycans was also decreased with thesupplementation of asparagine. The percentage of Mannoses in the controlsample (without asparagine addition) was as high as 10.6% (FIG. 38B). Inthe sample with the addition of asparagine the percentage of Mannoseswas decreased to 10.1% (0.4 g/L of asparagine), 8.3% (0.8 g/L ofasparagine), and 7.4% (1.6 g/L of asparagine), for a total reduction of3.2%.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. Furthermore, the strategies describedherein can be easily implemented either in-process or ad hoc to controlthe oligosaccharide distribution, thus reducing the potential impact ofraw material changes. For example, Adalimumab production strategies canuse these techniques to achieve maximized cell growth and specificproductivity without compromising product quality.

Patents, patent applications, publications, product descriptions,GenBank Accession Numbers, and protocols that may be cited throughoutthis application, the disclosures of which are incorporated herein byreference in their entireties for all purposes. For example, but not byway of limitation, patent applications designated by the followingattorney docket numbers are incorporated herein by reference in theirentireties for all purposes: 082254.0104; 082254.0235; 082254.0236;082254.0238; and 082254.0242.

We claim:
 1. A process for producing a recombinantly-expressedimmunoglobulin, comprising culturing a mammalian cell whichrecombinantly expresses the immunoglobulin during a production stage ina cell culture media comprising asparagine, thereby producing therecombinantly-expressed immunoglobulin, wherein the level of agalactosylfucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac)present on the produced immunoglobulin is increased as compared to thelevel of agalactosyl fucosylated biantennary oligosaccharides (sum ofNGA2F and NGA2F-GlcNac) of immunoglobulin produced in cell culture mediawhich does not comprise said asparagine during the production stage;and/or wherein the level of galactose containing fucosylated biantennaryoligossacharides (sum of NA1F and NA2F) present on the producedimmunoglobulin is decreased as compared to the level of galactosecontaining fucosylated biantennary oligossacharides (sum of NA1F andNA2F) of immunoglobulin produced in cell culture media which does notcomprise said asparagine during the production stage.
 2. The process ofclaim 1, wherein the immunoglobulin is an anti-TNFα antibody.
 3. Theprocess of claim 1, wherein the cell which expresses the immunoglobulinis a CHO cell.
 4. The process of claim 3, wherein the immunoglobulin isadalimumab.
 5. The process of claim 4, wherein the cell culture media(a) comprises asparagine at a concentration of between 0.4 g/L to 2.0g/L during the production stage; and/or (b) comprises asparagine at aconcentration of at least 0.4 g/L, at least 0.8 g/L, at least 1.2 g/L,at least 1.6 g/L or at least 2.0 g/L.
 6. The process of claim 4, whereinthe cell culture media (a) further comprises glutamine, optionally,wherein the cell culture media comprises glutamine at a concentration ofat least 0.4 g/L; (b) further comprises a yeast hydrolysate and/or aplant hydrolysate; optionally, wherein the yeast hydrolysate is selectedfrom the group consisting of Bacto TC Yeastolate, HyPep Yeast Extractand UF Yeast Hydrolysate; wherein the plant hydrolysate is selected fromthe group consisting of a soy hydrolysate, a wheat hydrolysate, a ricehydrolysate, a cotton seed hydrolysate, a pea hydrolysate, a cornhydrolysate, a potato hydrolysate, BBL Phytone Peptone, HyPep 1510, SE50MAF-UF, UF Soy Hydrolysate, Wheat Peptone E1, HyPep 4601 and ProyieldWGE80M Wheat; and/or wherein the yeast hydrolysate is present in thecell culture media at a concentration of between 2 g/L to 11 g/L and/orwherein the plant hydrolysate is present in the cell culture media at aconcentration of between 2 g/L to 15 g/L; and/or (c) is a chemicallydefined cell culture media.
 7. The process of claim 4, wherein the levelof agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2Fand NGA2F-GlcNAc) present on the produced immunoglobulin is 64%-88%,70%-88% or 75%-85%; and/or wherein the level of fucosylated biantennaryoligosaccharides (sum of NA1F and NA2F) present on the producedimmunoglobulin is 1%-30%, 2%-25%, 5%-20%, 5%-15%, 10%-20% or 27%-31%. 8.The process of claim 4, wherein the process is a fed batch process. 9.The process of claim 4, wherein the production stage initiates at aninitial viable cell density of approximately 0.5×10⁶ cells/mL.
 10. Theprocess of claim 1, further comprising collecting and isolating therecombinantly-expressed immunoglobulin.
 11. The process of claim 4,wherein the asparagine is present at a concentration of less than orequal to 26 mM.
 12. A process for producing a recombinantly-expressedimmunoglobulin, comprising culturing a mammalian cell whichrecombinantly expresses the immunoglobulin in a cell culture mediacomprising at least 0.8 g/L of asparagine, thereby producing therecombinantly-expressed immunoglobulin, wherein the level of agalactosylfucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac)present on the produced immunoglobulin is increased as compared to thelevel of agalactosyl fucosylated biantennary oligosaccharides (sum ofNGA2F and NGA2F-GlcNac) of immunoglobulin produced in cell culture mediawhich does not comprise said asparagine; and/or wherein the level ofgalactose containing fucosylated biantennary oligossacharides (sum ofNA1F and NA2F) present on the produced immunoglobulin is decreased ascompared to the level of galactose containing fucosylated biantennaryoligossacharides (sum of NA1F and NA2F) of immunoglobulin produced incell culture media which does not comprise said asparagine.
 13. Theprocess of claim 12, wherein the immunoglobulin is an anti-TNFαantibody.
 14. The process of claim 12, wherein the cell which expressesthe immunoglobulin is a CHO cell.
 15. The process of claim 14, whereinthe immunoglobulin is adalimumab.
 16. The process of claim 15, whereinthe cell culture media (a) comprises asparagine at a concentration of atleast 1.2 g/L, at least 1.6 g/L or at least 2.0 g/L; and/or (b)comprises asparagine at a concentration of between 0.8 g/L to 2.0 g/L.17. The process of claim 15, wherein the cell culture media (a) furthercomprises glutamine, optionally, wherein the cell culture mediacomprises glutamine at a concentration of at least 0.4 g/L; (b) furthercomprises a yeast hydrolysate and/or a plant hydrolysate; optionally,wherein the yeast hydrolysate is selected from the group consisting ofBacto TC Yeastolate, HyPep Yeast Extract and UF Yeast Hydrolysate;wherein the plant hydrolysate is selected from the group consisting of asoy hydrolysate, a wheat hydrolysate, a rice hydrolysate, a cotton seedhydrolysate, a pea hydrolysate, a corn hydrolysate, a potatohydrolysate, BBL Phytone Peptone, HyPep 1510, SE50 MAF-UF, UF SoyHydrolysate, Wheat Peptone E1, HyPep 4601 and Proyield WGE80M Wheat;and/or wherein the yeast hydrolysate is present in the cell culturemedia at a concentration of between 2 g/L to 11 g/L and/or wherein theplant hydrolysate is present in the cell culture media at aconcentration of between 2 g/L to 15 g/L; and/or (c) is a chemicallydefined cell culture media.
 18. The process of claim 15, wherein thelevel of agalactosyl fucosylated biantennary oligosaccharides (sum ofNGA2F and NGA2F-GlcNAc) present on the produced immunoglobulin is64%-88%, 70%-88% or 75%-85%; and/or wherein the level of fucosylatedbiantennary oligosaccharides (sum of NA1F and NA2F) present on theproduced immunoglobulin is 1%-30%, 2%-25%, 5%-20%, 5%-15%, 10%-20% or27%-31%.
 19. The process of claim 15, wherein the process is a fed batchprocess.
 20. The process of claim 12, further comprising collecting andisolating the recombinantly-expressed immunoglobulin.